The present invention relates to repression of gene expression. More specifically the invention relates to repression of -ene expression in plants by histone deacetylase, and histone deacetylase enzyme homologs.
Posttranslational modifications of histones in chromatin are important mechanisms in the regulation of gene expression. Acetylation of core histones is correlated with transcriptionally active chromatin of eukaryotic cells. Acetylation is thought to weaken the interactions of histones with DNA and induce alterations in nucleosome structure. These alterations enhance the accessibility of promoters to components of the transcription machinery, and increase transcription.
Histone deacetylation is thought to lead to a less accessible chromatin conformation, resulting in the repression of transcription (e.g. Pazin and Kadonaga, 1997; Struhl, 1998). The role of the yeast histone deacetylase, RPD3, in transcriptional repression was first discovered through a genetic screen for transcriptional repressors in S. cerevisiae (Vidal and Gaber, 1991). Since then, a number of yeast and mammalian histone deacetylase genes have been cloned (Rundlett et al., 1996; Emiliani et al., 1998; Hassig et al., 1998; Verdel and Khochbin, 1999). Most eukaryotic histone deacetylases show some sequence homology to yeast RPD3, suggesting that these proteins are all members derived from a single gene family (Khochbin and Wolffe, 1997; Verdel and Khochbin, 1999). In yeast and mammalian cells, the RPD3 histone deacetylases mediate transcriptional repression by interacting with specific DNA-binding proteins or associated corepressors and by recruitment to target promoters (Alland et al., 1997; Kadosh and Struhl, 1997; Hassig et al., 1997; Nagy et al., 1997; Gelmetti et al., 1998). Recently, a second family of histone deacetylases, HDA1 and related proteins, were identified in yeast and mammalian cells (Rundlett et al., 1996; Fischle et al., 1999; Verdel and Khochbin, 1999). The deacetylase domain of HDA 1-related proteins is homologous to but significantly different from that of RPD3 (Fischle et al. 1999; Verdel and Khochbin, 1999). These proteins also appear to be functionally different from RPD-like proteins in yeast cells (Rundlett et al., 1996). WO 97/35990 discloses mammalian-derived histone deacetylase (HD) gene sequences, gene products, and uses for these sequences and products. There is no disclosure of the use of these gene products for repressing gene expression.
In plants, an RPD3 homolog was first discovered in maize and it complemented the phenotype of a rpd3 null mutant of the yeast S. cerevisiae (Rossi et al, 1998). HD2 was also identified from maize that shows no sequence homology to yeast RPD3 or RPD3-related proteins (Lusser et al., 1997).
Even though histone deacetylation is thought to lead to repression of transcription, this has never been tested in plant systems. WO 98/48825 discloses the use of histone deacetylase (HD) for repressing gene expression in mammalian cell culture, however, the use of HD, or modified HD in plant gene repression is not disclosed. There is a plethora of information relating to the up-regulation of gene expression in plants, however, little is known on systems that can down regulate gene-expression. Thus, there is a need to develop regulatory systems for selectively repressing gene expression in plants.
The present invention pertains to novel histone deacetylase enzymes obtained from a plant. Four novel genes encoding histone deacetylases (AtRPD3A, AtRPD3B, AtHD2A and AtHD2B) and fragments thereof, were shown to be involved in the regulation of gene transcription within plants.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combination of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.
The present invention relates to repression of gene expression by histone deacetylase enzymes. More specifically the invention relates to repression of gene expression in plants by histone deacetylase enzymes.
According to the present invention there is provided a method of regulating gene expression in a transgenic plant comprising, introducing into a plant:
i) a first chimeric nucleotide sequence comprising a first regulatory element in operative association with a coding sequence of interest, and a controlling sequence; and
ii) a second chimeric nucleotide sequence comprising a second regulatory clement in operative association with a nucleotide sequence encoding histone deaceytlase and a nucleotide sequence encoding a DNA binding protein, the DNA binding protein having an affinity for the controlling sequence,
to produce the transgenic plant, and growing the transgenic plant.
The present invention is directed to the above method wherein the step of introducing comprises transforming the plant with the first, and the second, chimeric nucleotide sequence. Furthermore, the step of introducing comprises transforming a first plant with the first chimeric nucleotide sequence, and transforming a second plant with the second chimeric nucleotide sequence, followed by a step of crossing the first and the second plant, to produce the transgenic plant. Also included is the above method, wherein the step of introducing comprises transforming a plant with the first chimeric nucleotide sequence, followed by transforming the same plant with the second chimeric nucleotide sequence, or co-transforming a plant with both the first and second chimeric nucleotide sequences.
The present invention embraces the method as described above wherein the histone deacetylase, within the step of introducing, is selected from the group consisting of AtRPD3A, AtRPD3B, AtHD2A AtHD2B, an analogue, fragment, or derivative of AtRPD3A, AtRPD3B, AtHD2A AtHD2B, and a nucleotide sequence that hybridizes to AtRPD3A, AtRPD3B, AtHD2A AtHD2B at 65xc2x0 C. in 0.5 M Na2HPO4 (pH 7.2), 7% SDS, and 1 mM EDTA, wherein the analog, fragment, derivative, or nucleotide sequence that hybridizes encodes a product that exhibits repression of gene expression activity.
The present invention also relates to the method as described above wherein the upstream activating sequence and the DNA binding protein, within the step of introducing, are a Gal14 upstream activating sequence and a GAL4-binding protein, respectively. Furthermore, the first and the second regulatory region are selected from the group consisting of constitutive, tissue specific, developmentally-regulated, and inducible regulatory elements.
This invention is also directed to an isolated nucleotide sequence, selected from the group consisting of:
i) SEQ ID NO.:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7;
ii) an analog, derivative, fragment of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7; and
iii) a nucleotide sequence that hybridizes to SEQ ID NO:1, SEQ ED NO:3, SEQ ID NO:5, SEQ ID NO:7 at 65xc2x0 C. in 0.5 M Na2HPO4 (pH 7.2), 7% SDS, and 1 mM EDTA,
wherein the analog, derivative, fragment or the nucleotide sequence that hybridizes encodes a product that exhibits repression of gene expression activity. Furthermore, according to the present invention, there is also provided a chimeric construct comprising a regulatory element in operative association with the isolated nucleotide sequence as defined above, as well as a vector comprising the chimeric construct.
The present invention also pertains to an isolated amino acid sequence, selected from the group consisting of:
i) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8; and
ii) an analog, derivative, fragment of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
wherein the analog, derivative, or fragment exhibits repression of gene expression activity.
The present invention includes a transgenic plant, a transgenic plant cell, a transgenic seed, comprising said isolated nucleotide sequence as defined above.
The present invention is directed to a method of regulating Gene expression in a plant comprising:
i) introducing into the plant a chimeric nucleotide sequence comprising a regulatory element in operative association with a nucleotide sequence encoding, histone deaceytlase and a nucleotide sequence encoding a DNA binding protein, to produce a transgenic plant; and
ii) crowing the transgenic plant,
wherein the DNA binding protein has an affinity for a native controlling sequence within the plant.
The present invention also provides a method for altering a biochemical, physiological or developmental pathway of an organism comprising:
i) introducing into an organism a chimeric nucleotide sequence comprising a regulatory element in operative association with a nucleotide sequence encoding histone deaceytlase and a nucleotide sequence encoding a DNA binding protein specific for a controlling sequence; and
ii) growing the organism.
The present invention includes a method for identifying a DNA binding protein comprising:
i) introducing into a plant a chimeric nucleotide sequence comprising a regulatory element in operative association with a nucleotide sequence encoding histone deaceytlase fused with a nucleotide sequence of interest and of unknown function, to produce a transgenic plant;
ii) growing the transgenic plant; and
iii) examining the transgenic plant to determine whether the chimeric nucleotide sequence comprising the nucleotide sequence of interest has an effect on plant phenotype.
This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.